M1: Day 5 - Arsenic?

Introduction

You should define and understand the following vocabulary words. Ask your classmates or a TA if any of them are confusing to you.

Open Reading Frame (ORF)

Stop Codon

Primer

Template Strand

Coding Strand

Homology

Annealing

Designing Primers

You are probably familiar with the use of PCR reactions to selectively amplify a region of DNA, but you may not have considered what goes into designing a primer set. If you are amplifying a particular region of DNA, you may need to know the regions upstream and downstream of an open reading frame in order to identify regions where your primer can "touch down." These regions are called regions of homology, where a small stretch of genomic DNA serves as the template for a primer.

Finding a Target Sequence

Let's use the arsR gene as an example. We may know the sequence of this gene from either a literature search or from researching the registry of standard biological parts description. Note that the sequence listed for a std. biological part may be different than the original sequence. (This may be the result of point-mutations added to eliminate a restriction site or could be the product of codon optimization). In the case of the short arsR gene (518 bp), there are no point mutations so we can use the sequence directly as we search.

You can search by gene name or simply a sequence fragment to find what organisms might contain your gene of interest.
Use the Comprehensive Microbial Resource to see if the arsR gene is present in a wide range of organisms.
There is a "great deal to explore on this site, but you can navigate from the home page, by selecting the "Searches" tab. Select "find genes." In the search field type arsR and select "all genomes"

You could design a primer for a wide range of microbes, but you want to make sure the primer you design will work with one of the organism you have readily available to you. We have been using a strand of E.coli named K12-MG1655.

Use either the comprehensive microbial resource or a BLAST search to determine if E.coli MG1655 has the arsR gene.

What should be the target of your primer?

What sequence do you want to include in your final BioBrick part?

You should open a word file where you can copy and paste the sequences that you view online. This will help you later on.

Regions of Homology

There are multiple ways to do this next step. If you know another useful database, please feel free to share it with the class and your TAs.

Find the coordinate of your gene and associated region by searching for arsR

Search by "Gene Sym"

type: arsR

Record the coordinates of the gene. What do these coordinates reference? Will you need to see more or less sequence to design a primer for the part you wish to "harvest".

Go back to Mg1655 genome page and use "Gene Retrieval by Coordinates"

Type coordinates you wish to view.

Copy this sequence into a word document

Identify the beginning of the arsR open reading frame and bold it

Identify the end of arsR open reading frame and bold it

Identify the arsR operator and sequence where the protein binds to repress transcription

In different colors indicate the regions of homology for your forward primer and reverse primer

Most oligonucleotide primers tend to have a region of homology with the template stand of 18-20 nucleotides in length. You may include an additional overhang on the 5' end of your primer, but you will need to select 18-20 nucleotides that match the genome you are trying to amplify.

Select a region of homology in the forward direction

Select a region of homology in the backwards direction.

Standard Assembly Parts

On the 5' ends of each of your primers you now need to ad overhangs that will introduce the restriction sites to the arsR functional component of the part.

Sketch a diagram of the final part you want to create including the restriction sites

Design a forward primer

Design a reverse primer

Make sure your final primers includes both an overhang and a 18-20bp region of homology with the MG1655 sequence

REMEMBER: WHEN YOU ORDER PRIMERS YOU ALWAYS WRITE THE SEQUENCE IN THE 5' to 3' DIRECTION

Testing the Annealing Temperature

Primers anneal and separate from their template strand at different temperatures. A good primer set should have a similar annealing temperature for both te forward and reverse primers. When thinking about annealing temperature, you only need to concern yourself with the annealing temperature of the homologous (18-20bp) regions, not the whole primer sequence.

What do you think governs annealing temperature?

Test and record the annealing temperature of your primers at this handy site:

What else might you be concerned with when designing a primer?

Ordering a Primer

Once you have shown a TA your design and received the go ahead, you can test your primer design by entering the sequence you wish to amplify into a BioBricks standard part generator at this website: (Don't do this until your primer design is complete or you will receive no credit for today's lab): Primer Designer

In Class

In class today you should accomplish four things:

Design primers.

Set up your colony PCR.

Mini-prep your vector.

Set up a digest of your vector.

Colony PCR

Materials

10x TAQ buffer

25mM MgCl2

dNTPs

forward primer (10uM)

reverse primer (10uM)

sterile water

TAQ polymerase

plate with MG1655

Protocol

Add the following items to a PCR tube:

73uL water

10uL 10x TAQ buffer

10uL 25mM MgCl2

2uL dNTPs

2uL forward primer

2uL reverse primer

1uL TAQ polymerase

Pick a single colony from your MG1655 plate with a pipette tip and swirl it in your PCR mix.

Enter in the following program on the PCR block (What do you think each of these steps does?):

Cycles

Temp

Time

1x

95C

10 min

30x

94C

45s

55C

30s

72C

1min 20s

1x

15C

forever

Put your sample in the PCR block and run the program! It should take about 2 hours.

In the mean time....

Reporter Selection and Digestion

Once you have isolated the arsenic sensor, you'll drop it in front of a reporter - in this case our color generators. This time you get to choose which color generator you would like to use.

Protocol

Cultures have been inoculated for you that contain each of the color generators (with no promoters). Pick one and mini-prep it to isolate your plasmid. Measure the concentration.

Decide what restriction enzymes you will use to digest it.

Set up the restriction digest. We will gel extract these next Tuesday.